A microgel assembly scaffold capable of promoting skin wound healing and a preparation method thereof

Abstract

The present invention discloses a microgel assembly scaffold that can promote skin wound healing and a preparation method thereof. The preparation method of the microgel assembly scaffold is as follows: hydrogel microspheres modified with bisphosphonic acid groups and hydroxyapatite nanoparticles doped with zinc ions are synthesized separately, the bisphosphonic acid groups in the microgels and the zinc ions in the nanoparticles produce a strong coordination effect, and the nanoparticles are used to assemble the microgels into one body to form a porous microgel assembly scaffold. The above-mentioned microgel assembly scheme can efficiently and quickly realize the assembly between hydrogel microspheres, and avoids the biological toxicity caused by the use of free zinc ions. The hydrogel assembly scaffold formed by this assembly scheme has porosity, injectability, self-healing properties, morphological adaptability, macroscopic mechanical properties and excellent biocompatibility, and can be applied to the field of tissue regeneration, especially for the treatment of irregular tissue wounds, to promote tissue repair and regeneration.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomaterials, tissue engineering and regenerative medicine, and particularly relates to a microgel assembly scaffold capable of promoting skin wound healing and a preparation method thereof. Background Art

[0002] Normal wound healing is divided into four synchronous processes: hemostasis, inflammation, proliferation, and remodeling. The proliferation phase involves the formation of granulation tissue composed of fibroblasts, myofibroblasts, and keratinocytes. During this process, fibroblasts and myofibroblasts proliferate excessively, accompanied by an imbalance in the synthesis and degradation of collagen, leading to excessive fibrosis, which is the scarring of the wound. Hydrogels are a biomaterial with adjustable mechanical properties, good biocompatibility, high water content, and high water absorption. The dressings made from them can quickly absorb wound exudate and inhibit bacterial infection. They are a commonly used material in skin tissue repair. An ideal wound dressing should have good biocompatibility, exudate absorption and moisture retention, a certain mechanical strength, breathability, etc. However, traditional hydrogel dressings usually find it difficult to balance the breathability and exudate absorption capabilities of the gel, and cannot support the penetration of cells into the gel. Therefore, it is of great significance to study a new type of hydrogel dressing based on microgel assembly.

[0003] Porous hydrogel scaffolds assembled from microgels for skin wound healing balance water absorption and air permeability, and their porous structure facilitates cell infiltration into the gel, enabling endogenous repair. Modular combinations of microgels with different components and functions can achieve multiple therapeutic benefits, including anti-inflammatory, antioxidant, and antibacterial properties. By improving the air permeability and cell infiltration properties of hydrogel dressings, while imparting anti-inflammatory and drug delivery properties, porous microgel scaffolds can play a role in accelerating wound healing and reducing scarring.

[0004] Microgels are hydrogels ranging in size from a few micrometers to several hundred micrometers. Their large surface area facilitates the absorption of wound exudate. Microgel assembly can be used to create larger microgel scaffolds, expanding their applications in various fields. Microgel assembly methods include chemical reaction assembly, physical action assembly, and cell-mediated microgel assembly. Chemical reaction assembly can produce microgel scaffolds with good shape stability, but their flowability and injectability are extremely poor. Cell-mediated microgel assembly is time-consuming and has limited assembly strength. Physically assembled microgel assemblies can achieve both injectability and mechanical properties and are currently a hot topic of research. Commonly used assembly forces include host-guest interactions, electrostatic interactions, hydrogen bonding, and biotin-avidin interactions. Existing physical forces can generally achieve simple stacking and assembly of hydrogel particles, but these forces are not strong enough, which can lead to poor mechanical properties of the microgel assemblies, resulting in scattered and fluid microgels, which is detrimental to wound healing. The coordination effect of metal ions is a very strong physical effect. For example, the binding constant of zinc ions to bisphosphonic acid is 10 29 This force can significantly address the issue of decreased mechanical properties after microgel physical assembly. Furthermore, based on the theory of multiple bonding, constructing microgel assemblies through multiple physical bonding interactions, such as hydrogen bonds, coordination bonds, and classical interactions, can simultaneously impart excellent mechanical properties, facilitating their application as skin wound dressings.

[0005] However, the direct use of metal ions will bring about obvious biological toxicity and is not suitable for the biomedical field. Zinc ions have concentration-dependent effects on cell viability, cell proliferation, vascularization, coagulation, osteogenesis and other processes: Existing studies have shown that when the zinc ion concentration is greater than 100μM, it generally has obvious cytotoxicity to endothelial cells, smooth muscle cells, fibroblasts and other cells, thereby inhibiting cell proliferation; at lower concentrations (below 80μM), zinc ions have a promoting effect on cell adhesion, proliferation and migration. Hydroxyapatite doped with zinc ions is an inorganic material with excellent biocompatibility. Zinc ions are slowly dissolved and released, which can greatly reduce the local zinc ion concentration and improve cell and blood compatibility. It also has bioactive functions such as degradability and antibacterial properties. Therefore, compared with the direct use of zinc ions, zinc-doped hydroxyapatite has low biotoxicity and other bioactive functions, and is more suitable for the field of tissue regeneration. Summary of the Invention

[0006] To address the challenges of existing hydrogel dressings in achieving both porosity, self-healing properties, and mechanical properties, as well as the insufficient assembly strength and poor biocompatibility of microgel assembly scaffolds, the present invention provides a microgel assembly scaffold capable of promoting skin wound healing and a method for preparing the same. This approach utilizes the strong synergistic effect between the bisphosphonate groups in the microgel and the zinc ions in the nanoparticles to assemble microgel microspheres into a granular microgel scaffold. This microgel assembly scheme allows for efficient and rapid assembly between hydrogel microspheres while avoiding the biotoxicity of direct zinc ion use. The resulting hydrogel assembly scaffold exhibits porosity, injectability, self-healing properties, macroscopic mechanical properties, and excellent biocompatibility.

[0007] In order to solve the related problems in the technical background, the present invention adopts the following technical solutions:

[0008] Bisphosphonate-modified hydrogel microspheres and zinc-doped hydroxyapatite nanoparticles were prepared. The modified hydrogel suspension and the modified nanoparticle suspension were thoroughly mixed, and microgel assembly was achieved through the strong coordination between the bisphosphonate groups in the microgels and the zinc ions in the nanoparticles.

[0009] Furthermore, the specific steps for preparing hydrogel microspheres modified with bisphosphonic acid groups are as follows: (1) using methacrylic anhydride to modify compound A to prepare a polymer monomer with a bisphosphonic acid structure; wherein compound A includes sodium alendronate, sodium pamidronate, sodium hydroxyethyl diphosphate, etc.; (2) introducing the bisphosphonic acid monomer into the hydrogel prepolymer solution, preparing microgels in a microfluidic device, and cross-linking and curing the microgels under ultraviolet light.

[0010] Furthermore, the preparation process of a polymeric monomer with a bisphosphonic acid structure is as follows: Compound A is dissolved in water, the pH is adjusted to 7-8 with NaOH, methacrylic anhydride is added dropwise, and the reaction is carried out in an ice bath under nitrogen protection for at least 8 hours. After the reaction is completed, post-processing steps such as concentration, precipitation, recrystallization, and freeze-drying are performed to obtain a pure white solid, which is the bisphosphonic acid monomer.

[0011] Furthermore, the hydrogel is prepared by ultraviolet cross-linking. The hydrogel prepolymer solution is composed of a bisphosphonic acid monomer, other monomers or macromolecules, and a photoinitiator. The other monomers or macromolecules include one or more of acrylic acid, acrylamide, methacrylated gelatin, and methacrylated hyaluronic acid. The lithium phenyl-2,4,6-trimethylbenzoylphosphonate in the hydrogel prepolymer solution acts as a photoinitiator and can initiate cross-linking under ultraviolet light.

[0012] Furthermore, microgels were prepared using a microfluidic device with the aforementioned hydrogel prepolymer as the aqueous phase and paraffin oil containing 15% by volume of a Span-80 surfactant as the oil phase. Hydrogel microspheres of varying sizes were prepared by adjusting the flow rates of the aqueous and oil phases. The aqueous phase flow rate was 650 μL / h, and the oil phase flow rate varied from 1 to 4 mL / h.

[0013] Furthermore, zinc ion-doped hydroxyapatite nanoparticles can be prepared by a co-precipitation method, with the zinc ion doping molar amount being 5-15%.

[0014] Furthermore, the microgel assembly protocol is as follows: the microgels are suspended in PBS to prepare a 100 mg / mL microgel suspension, designated as Solution A. Zinc-doped hydroxyapatite nanoparticles are prepared into a 50 mg / mL suspension, designated as Solution B. 1 mL of Solution A and 40-200 μL of Solution B are mixed and thoroughly vortexed. The microgels self-assemble under the action of the nanoparticles, resulting in a microgel assembly scaffold.

[0015] By the above preparation method, a microgel assembly scaffold that can be used to promote skin wound healing is obtained, which can be used to prepare a dressing or a medicament that promotes skin wound healing.

[0016] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0017] This invention provides a novel approach and strategy for microgel assembly, featuring a simple reaction process, rapid and easy assembly, strong assembly force, and avoiding the biotoxicity of direct zinc ion use. The resulting assembled scaffold exhibits the following advantages: ① injectability and self-healing properties; ② porosity and breathability; ③ moderate and adjustable mechanical properties; and ④ biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of microgel assembly mediated by bisphosphonates and zinc nanoparticles

[0019] Figure 2 Structural characterization of bisphosphonic acid monomers

[0020] Figure 3 The rheological behavior test results of the microgel assembly scaffolds obtained in Examples 1-3 and Comparative Example 1 are shown in FIG.

[0021] Figure 4 Evaluation of the blood compatibility of the microgel assembly scaffolds obtained in Examples 1-3 and Comparative Example 1

[0022] Figure 5 Evaluation of the cell compatibility of the microgel assembly scaffolds obtained in Examples 1-3 and Comparative Examples 1 and 2

[0023] Figure 6 This is a macroscopic morphology photo of the assembly bracket of Example 3

[0024] Figure 7 Verification of the injectability and self-healing properties of the microgel assembly scaffold obtained in Example 3

[0025] Figure 8 Porosity characterization of the microgel-assembled scaffold obtained in Example 3

[0026] Figure 9 Macroscopic compression characterization of the microgel-assembled scaffolds obtained in Example 3 and Comparative Example 1 Specific implementation plan

[0027] The technical solution of the present invention is further described below through specific embodiments and drawings.

[0028] The following is a detailed description of the synthesis steps of the bisphosphonic acid monomer, taking methacrylic anhydride modified alendronate as an example. Sodium alendronate trihydrate is dissolved in deionized water, NaOH is added to adjust the pH to 7-8, and an excess of methacrylic anhydride is added dropwise in an ice bath and under nitrogen protection. The reaction is carried out in an ice bath for 8 hours, and the pH is continuously adjusted to a weak alkaline state during the reaction. After the reaction is completed, it is acidified with dilute hydrochloric acid, concentrated by rotary evaporation, and precipitated with acetone and ethanol to obtain a crude product. After multiple recrystallizations, pure methacrylated alendronate is obtained, which is the bisphosphonic acid monomer (structure as shown in FIG. Figure 2 ). To specifically illustrate the beneficial effects of the present invention, the prepolymer liquid of the hydrogel in the following examples and comparative examples is composed of a bisphosphonic acid monomer, acrylamide, a crosslinker and an initiator. The crosslinker used is methacrylated gelatin, and the ultraviolet light initiator used is lithium phenyl-2,4,6-trimethylbenzoylphosphonate. The zinc ion-doped hydroxyapatite nanoparticles are prepared by a hydrothermal method, wherein calcium salt, zinc salt and phosphate are dissolved in water according to a proportion, a precipitant ammonia water is added to adjust the pH to 10, the mixture is stirred and reacted at 120°C overnight; the obtained precipitate is washed multiple times in deionized water and dried to obtain zinc particle-doped hydroxyapatite nanoparticles. It is only used to explain the technical scheme and effects of the present invention in combination with the following examples, and is not used to limit the scope of protection of the present invention. The samples of different embodiments and comparative examples are described in detail below.

[0029] Example 1

[0030] A hydrogel prepolymer solution was prepared by mixing acrylamide, bisphosphonic acid monomer, methacrylated gelatin, and lithium phenyl-2,4,6-trimethylbenzoylphosphonate at 25%, 5%, 2.5%, and 0.1% by weight, respectively. Microgels were prepared using a microfluidic device with the prepolymer solution as the aqueous phase and paraffin oil containing 15% Span-80 surfactant as the oil phase. The aqueous phase flow rate was 650 μL / h, and the oil phase flow rate was 1.5 mL / h. The resulting microgels had a size of 400 μm.

[0031] The preparation method of the microgel assembly scaffold of this embodiment is as follows: the microgel is suspended in PBS solution to prepare a 100 mg / mL microgel suspension, which is recorded as liquid A. Hydroxyapatite nanoparticles doped with 10 mol% zinc ions are prepared into a 50 mg / mL suspension, which is recorded as liquid B. 1 mL of liquid A and 25 μL of liquid B are mixed and vortexed thoroughly. The microgels assemble themselves under the action of the nanoparticles to obtain a microgel assembly scaffold. Assembly process diagram Figure 1 .

[0032] Rheological behavior test of the assembled scaffold is shown in Figure 3 The storage modulus of the assembled scaffold is 421.16±100.83Pa, and the loss modulus is 68.67±54.65Pa. The storage modulus is much larger than the loss modulus, which proves that the material has a large solidity when it is static, indicating the successful construction of the assembled scaffold. The biocompatibility test of the assembled scaffold is shown in Figure 4 and Figure 5 The cell compatibility was tested using the CCK-8 method, and the cell viability was 107.97±7.55%, with almost no hemolysis. It can be seen that the material has excellent cell compatibility and blood compatibility.

[0033] Example 2

[0034] The volume of liquid B used in the assembly process was 50 μL, and the rest was the same as in Example 1. The rheological behavior test of the assembled scaffold of this example is shown in Figure 3 The storage modulus of the assembled scaffold is 1740.27±43.55Pa, and the loss modulus is 106.87±16.52Pa. The storage modulus is much larger than the loss modulus, which proves that the material has a large solidity when it is static and is not easy to flow. The storage modulus of the assembled scaffold can be regulated by adjusting the content of liquid B. That is, as the amount of doped zinc hydroxyapatite increases, a stronger interaction is formed. The scaffold has a larger storage modulus than Example 1, which means that the interface interaction of the assembled scaffold formed by it is more reliable. The biocompatibility test of the prepared assembled scaffold is shown in Figure 4 and Figure 5 The cell viability was 104.18±4.52, and almost no hemolysis occurred, indicating that the material has excellent cell compatibility and blood compatibility.

[0035] Example 3

[0036] The volume of liquid B used in the assembly process was 75 μL, and the rest was the same as in Example 1. The macroscopic photograph of the assembled stent of this example is shown in FIG. Figure 6 The sheet-like assembly scaffold can be picked up with tweezers, and the columnar assembly scaffold can support its own weight, indicating that the microgel assembly scaffold has a reliable and strong interface interaction. The rheological behavior test of the assembly scaffold is shown in Figure 3 The storage modulus of the assembled scaffold is 2540.99±24.62Pa, and the loss modulus is 194.37±25.87Pa. The storage modulus is much larger than the loss modulus, which proves that the material has a large solidity when it is static and is not easy to flow. The storage modulus of the assembled scaffold in this embodiment is significantly improved compared with that of Examples 1 and 2, which also proves that the interface effect is more reliable and forms a more reliable and balanced interface interaction. Its injectability and self-healing properties are verified in Figure 7 , 5% and 500% oscillatory strains were applied to the assembled scaffold. It can be seen that the material remains solid at 5% strain and undergoes shear thinning at 500% strain, showing fluidity, indicating that it is injectable. After multiple recovery strains to 5%, the modulus of the material does not change significantly, indicating that it has self-healing properties. Its biocompatibility test is shown in Figure 4 and Figure 5 The cell viability was 99.49±7.43, and almost no hemolysis occurred, indicating that the material has excellent cell compatibility and blood compatibility.

[0037] The porosity statistics are shown in Figure 8 The porosity of the microgel assembly scaffold formed after vacuum filtration was 27.66±16.75%.

[0038] Its macroscopic mechanical properties (compression properties) are shown in Figure 9 After nanoparticle assembly, the compressive stress-strain and compression modulus are significantly improved, indicating that the assembled scaffold has a macroscopic mechanical strength similar to that of bulk gel.

[0039] Comparative Example 1

[0040] The volume of solution B used in the assembly process was 0 μL, and the rest was the same as in Example 1.

[0041] Comparative Example 1 Rheological behavior test of the assembled support Figure 3 The storage modulus of the assembled support is 295.84±0.77Pa, and the loss modulus is 8.88±1.04Pa. Its storage modulus is significantly smaller than that of the materials in Examples 1, 2, and 3. The compressive stress strain and compression modulus test of the assembled support in Comparative Example 1 are shown in FIG. Figure 9The macroscopic mechanical properties of the material that has not been assembled with nanoparticles are poor. In summary, the material in Comparative Example 1 does not form effective assembly between microgels.

[0042] The biocompatibility test of the material of comparative example 1 is shown in Figure 4 and Figure 5 , the cell viability was 103.46±6.56%, and almost no hemolysis occurred, indicating that the material has excellent cell compatibility and blood compatibility.

[0043] Comparative Example 2

[0044] During the assembly process, liquid B was changed to 0.1 mol / L ZnCl2 solution, and the rest was the same as in Example 1. Cell compatibility test of materials in Comparative Example 2 is shown in Figure 4 , the cell viability was 76.40±3.83%. Compared with Examples 1-3, it is obvious that the use of zinc-containing nanoparticles in the present invention has lower cytotoxicity and is safer and more reliable than the direct use of zinc ions in this example.

Claims

1. A method for preparing a microgel assembly scaffold that can promote skin wound healing, characterized in that: First, hydrogel microspheres modified with bisphosphonic acid groups and hydroxyapatite nanoparticles doped with zinc ions are prepared, and modified hydrogel suspensions and modified nanoparticle suspensions are obtained using the microspheres and nanoparticles, respectively. The two suspensions are then fully mixed, and the microgel microspheres are assembled into a granular microgel scaffold through the coordination of the bisphosphonic acid groups in the microgel and the zinc ions in the nanoparticles. The specific steps of preparing hydrogel microspheres modified with bisphosphonic acid groups include: (1) modifying compound A with methacrylic anhydride to prepare a polymer monomer with a bisphosphonic acid structure; wherein compound A is one or more of alendronate sodium, pamidronate sodium, and hydroxyethyl diphosphate sodium; (2) introducing the polymer monomer with a bisphosphonic acid structure into a hydrogel prepolymer solution, preparing microgels in a microfluidic device, and cross-linking and curing the microgels under ultraviolet light to obtain the hydrogel microspheres; The hydrogel microspheres were suspended in PBS solution to prepare a 100 mg / mL microgel suspension, recorded as liquid A. The zinc ion-doped hydroxyapatite nanoparticles were prepared into a 50 mg / mL suspension with deionized water, recorded as liquid B. Liquid A and liquid B were mixed at a volume ratio of 1 mL:40-200 μL and vortexed thoroughly. The microgels assembled themselves under the action of the nanoparticles and were collected by filtration to obtain a microgel assembly scaffold with a porosity of 20-30%.

2. The preparation method according to claim 1, characterized in that The step (1) specifically comprises: dissolving compound A in water, adjusting the pH to 7-8 with NaOH, adding methacrylic anhydride dropwise, reacting for more than 8 hours under ice bath and nitrogen protection, and after the reaction, performing post-treatment steps of acidification, concentration, precipitation, and freeze-drying to obtain a pure white solid, which is a polymer monomer with a bisphosphonic acid structure.

3. The preparation method according to claim 1, characterized in that The hydrogel prepolymer solution includes the polymerizable monomer with a bisphosphonic acid structure, other monomers or macromolecules, and a photoinitiator. The other monomers or macromolecules are one or more of acrylic acid, acrylamide, methacrylated gelatin, and methacrylated hyaluronic acid. Lithium phenyl-2,4,6-trimethylbenzoylphosphonate is used as the photoinitiator.

4. The preparation method according to claim 1, wherein Microgels were prepared using a microfluidic device. The aqueous phase was the hydrogel prepolymer solution, and the oil phase was paraffin oil containing 15% by volume of Span-80 surfactant. By adjusting the flow rates of the water and oil phases, hydrogel microspheres with a size of 100-500 μm were prepared, wherein the aqueous phase flow rate was 650 μL / h and the oil phase flow rate was 1-4 mL / h.

5. The preparation method according to claim 1, wherein The zinc ion-doped hydroxyapatite nanoparticles are prepared by a hydrothermal method, and the zinc ion doping amount is 5-15% by molar ratio.

6. A microgel assembly scaffold, characterized in that: The stent is prepared by the preparation method according to any one of claims 1 to 5.

7. The use of the microgel assembly scaffold according to claim 6, characterized in that: The microgel assembly scaffold is used for preparing a dressing or a medicament for promoting skin wound healing.

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